Method for preparing stabilized prostaglandin E1

ABSTRACT

A stable and lyophilized formulation of prostaglandin E-1 made by the process comprising a) dissolving PGE-1 in a solution of lactose and tertiary butyl alcohol wherein said tertiary butyl alcohol is present in an amount of from about 15% to about 33% volume/volume and the ratio of said lactose to PGE-1 is from about 40,000 to 1 to about 10,000 to 1 weight/weight whereby a formulation of PGE-1 dispersed in lactose is formed; b) adjusting and maintaining the pH of said formulation from about 3.5 to about 6 with an organic acid buffer, c) freezing said formulation to about -50° C.; and d) drying said formulation to obtain a moisture content of less than 1% by dry weight and a tertiary butyl alcohol content of less than 3% by dry weight. Preferably, step c) includes after freezing said formulation to about -50° C., warming to about -25° C. for about 2 hours then refreezing to about -50° C. Preferably, the prostaglandin is in an amount of about 25 to 100 ppm in lactose and the pH is maintained at about 4 to 5.

This application is a divisional of U.S. Ser. No. 08/619,690, filed Mar.28, 1996, pending which was the national phase of PCT/US94/109648 filedSep. 6, 1994 which was a continuation-in-part of U.S. Ser. No.08/143,695, filed Oct. 27, 1993, abandoned.

BACKGROUND OF THE INVENTION

Prostaglandin E₁ (PGE-1) is an inherently unstable compound. PGE-1 ischemically (11α, 13E, 15S11,15-dihydroxy-9-oxoprost-13-en-1-oic acid; or3-hydroxy-2-(3-hydroxy-1-octenyl)-5-oxo-cyclopentaneheptanoic acid; andis commonly referred to as alprostadil or PGE₁. PGE-1 is a primaryprostaglandin which is easily crystallized from purified biologicalextracts. A goal of this invention was the development of a roomtemperature stable formulation of PGE-1. More preferred would be amethod to stabilize a low dose (5-20 μg) formulation of PGE-1 suitablefor use in the treatment of erectile dysfunction.

Various attempts to freeze dry PGE-1 have been described in U.S. Pat.Nos. 3,952,004 and 3,927,197. The first patent "004 describes thestabilization of PGE-1 in tertiary butyl alcohol and sodium chloride andthat lactose or other "simple sugars" destabilize PGE-1. The secondpatent '197 describes PGE-1 stabilized with tertiary butyl alcohol.

Despite various attempts to stabilize PGE-1, better and more effectivemethods are in demand to increase shelf life and maintain efficacy.PGE-1 formulations in lactose appears to degrade through an apparentsecond order mechanism with respect to PGE₁ concentration in the solidstate. Maximum stability can be achieved by either minimizing the PGE-1concentration in a suitable lactose diluent or by optimizing otherparameters which may impact the second order rate constant. The secondorder rate constant is affected by the solid state pH, the buffercontent, the moisture content, the use of tertiary butyl alcohol duringprocessing, the freezing rate, and the drying rate. All of theseparameters have been optimized to minimize the value of the second orderrate constant.

SUMMARY OF THE INVENTION

In one aspect the subject invention is a lyophilized formulation ofPGE-1 made by the process comprising:

a) dissolving PGE-1 in a solution of lactose and tertiary butyl alcoholwherein the tertiary butyl alcohol is present in an amount of from about15% to 33% volume/volume and the ratio of lactose to PGE-1 is from about40,000 to 1 to about 10,000 to 1 weight/weight (25 to 100 ppm inlactose) whereby a formulation of PGE-1dispersed in lactose is formed;

b) adjusting and maintaining the pH of the formulation from about 3.5 toabout 6 with an organic acid buffer (preferably sodium citrate);

c) freezing the formulation to about -50° C.; and

d) drying the formulation to obtain a moisture content of less than 1%by dry weight and a tertiary butyl alcohol content of less than 3% bydry weight. Preferably, the PGE-1 in the formulation is in an amount ofabout 25 to 100 ppm lactose and the pH is adjusted and maintained at 4to 5 by the presence of a buffer. Preferably, the freezing step (c)includes an annealing process by freezing the formulation to about -50°C., warming to about -25° C. for about 2 hours then refreezing to about-50° C.

In another aspect, the subject invention is a method for preparing astabilized, lyophilized formulation of PGE-1 comprising the steps setforth above.

In yet another aspect, the present invention is a freeze-driedformulation of PGE-1 for use in the treatment of erectile dysfunction.Typical dosages of the formulation are (5-20 μg) formulations of PGE-1.These formulations correspond to a ratio of lactose to PGE-1 of fromabout 40,000 to 1 to about 10,000 to 1 weight/weight (25 to 100 ppm inlactose). That is, 5 μg corresponds to 40,000 to 1; 10 μg corresponds to20,000 to 1 and 20 μg corresponds to 10,000 to 1.

DETAILED DESCRIPTION OR THE INVENTION

The subject invention is a lyophilized PGE-1 composition made from abulk sterile filtered solution which contains 20% v/v tertiary butylalcohol (TBA) and has an apparent pH of approximately 4. Both the waterand TBA are removed during the freeze-dying process. Residual water andTBA remaining after lyophilization are<0.5% and 0.5-2% respectively ofthe dried cake mass. In one formulation, a vial dosage contains aftercompletion of lyophilization: 23 μg of PGE-1 (alprostadil), 193.8 mg ofanhydrous lactose, and 53 μg of sodium citrate. After reconstitution ofthis freeze-dried powder with 1.0 ml of either water for injection orbacteriostatic water for injection, a solution containing 20 μg/ml ofPGE-1 is obtained. The freeze-dried powder is packaged in a 5 ml vialand sealed with a lyophilization style closure within the freeze-drychamber, and capped with an aluminum overseal. The chemical stability ofthe PGE-1 can be predicted by use of the Arrhenius equation andaccelerated stability data. Initial rate kinetic analyses (i.e.,monitoring the rate of formation of the major degradation product, PGA₁)can be used to assess the chemical stability. The projected stabilityanalysis indicates that when the product is properly manufactured withthe optimized formulation and process, the shelf-life should be greaterthan 24 months when the product is stored at 25° C. or less.

Initial work centered on the use of a lactose diluent and lyophilizationfrom a tertiary butyl alcohol (TBA)/water co-solvent system. Thefreeze-dried formulation produced appeared to possess the properties ofa solid solution. The degradation of the PGE-1 in this type offormulation could be best described by a second order mechanism.Stability could be increased by maximizing the amount of lactose diluentor minimizing the amount of PGE-1 present. The solid state second orderkinetics fit well to an Arrhenius type temperature relationship.Residual moisture was shown to have a deleterious effect on thestability. The pH of the cake also affected the stability. Optimumstability was achieved at about pH 4-5. A minimum amount of citratebuffer was added to the formula to control pH. Lyophilization from aTBA/water co-solvent mixture improved stability of the formulationscompared to water only. Typically, standard freeze drying techniques canbe used to prepare the stabilized PGE-1. More preferably, an annealingtechnique can be performed to decrease and more uniformly control theresidual tertiary butyl alcohol in the freeze dried product. Optimumstability was achieved when freeze-drying from a 17-25% TBA/watermixture.

The method for preparing a stabilized, freeze-dried formulation of PGE-1controls key parameters which affect product stability including thefollowing: the level of lactose diluent present, the apparent pH of thelyophilized cake, the moisture content, the use of the co-solventtertiary butyl alcohol during processing, the freezing rate andmethodology prior to lyophilization, the freeze-drying rate, and thesize of the vial used to manufacture the product.

Lyophilization of a buffered lactose formulation of PGE-1 from atertiary butyl alcohol (TBA)/water mixture provides superior productstability than when freeze-drying from only an aqueous system. The levelof TBA which afforded the product maximum stability appeared to be whenthe TBA amount ranged from 17-25% (v/v). The 20% TBA level was selectedas the amount of co-solvent for the PGE-1 formulation since it fellwithin the optimum co-solvent range. The lower the level of TBA usedalso reduced the flammability potential, reduced the amount of TBA wastewhich would be generated after lyophilization, reduced the level ofisopropyl alcohol (a process impurity in the TBA) in the product, andlowered the precipitation potential during manufacturing for the lactosefrom the co-solvent system. Stability data clearly indicated thatlyophilization of the buffered lactose formulation of PGE-1 from aTBA/water co-solvent system would be significantly more stable thanfreeze-drying from an aqueous system. The mechanism for the improvementin stability when using the TBA is unknown but it is likely enabling thePGE-1 molecules to be kept further apart during the freezing andlyophilization phases of manufacture. Therefore, it is recommended thatthe final formulation be lyophilized from a co-solvent system containing20% v/v TBA It is important that this level of TBA be used because lowerlevels of TBA (≦17%) in the final formulation will produce a productwhich is much less stable than when using at least 20% TBA

The resulting residual TBA in the final product is expected to beapproximately 0.5 to 2% of the cake weight. A safety assessment of TBAand its impurities (isopropyl alcohol, 2-butyl alcohol, and isobutylalcohol) concluded that TBA levels of not more than 3% of a 200 mgfreeze-dried cake was acceptable and that other residual organicsolvents should be not more than 0.5% of a 200 mg freeze-dried cake.

The application of heat to the lactose may adversely affect productstability. The stability data clearly dictates that close control of thefreeze-dry cycle (both primary and secondary drying) is critical toreproducible manufacture lots with equivalent stability. Stability cantherefore be a function of processing parameters.

Since the PGE-1 degradation kinetics fit (at least empirically) a secondorder mechanism, stability can be improved by simple dilution of thePGE-1 with lactose. This would suggest that maximizing the amount oflactose for a given amount of PGE-1 should provide the optimumstability. For a 20 μg/ml formulation of PGE-1 the amount of lactosechosen for formulating the optimum formulation is 204 mg of lactosemonohydrate. After lyophilization, the 5% water is removed and theresulting lactose present in the vial is 193.8 mg. The cake volume for193.8 mg of anhydrous lactose is approximately 0.13 ml. Therefore, thetheoretical solution concentration of lactose after reconstitution with1.0 ml is (193.8 mg/1.13 ml) or 172 mg/ml. The amount of PGE-1 needed toproduce a 20 μg/ml solution after reconstitution with 1.0 ml is (1.13ml×20 μg/ml) or 22.6 μg.

It has been determined that the cake pH also affected product stability.Maximum stability is achieved when the cake pH is held near pH 4 to 5.Both citrate and acetate buffers can be used; however, citrate bufferwas selected as the buffer of choice for the PGE-1 formula since it is acommon buffer for parenteral products. Since PGE-1 is susceptible toboth acid and base hydrolysis, it is probable that some buffer catalysisof the PGE-1 molecule may occur. The amount of citrate buffer selectedfor the final formulation was chosen based on a compromise betweensufficient buffer to adequately control pH and yet not itselfsignificantly provide an alternate catalytic route. The level of citratechosen for the final formulation was 53 μg of sodium citrate/23 μg PGE-1(3.17 moles citrate/mole PGE-1). This level of citrate willtheoretically cause only a relatively minor increase (<7%) in thedegradation rate constant. In order to determine if sufficient bufferwas present to control pH for the shelf life of the product, sampleswith this amount of buffer present were degraded to less than 90% ofinitial potency under accelerated conditions. The pH was measuredinitially and after the>10% drop in potency had taken place. Nosignificant change in pH occurred. This demonstrates that sufficientbuffer was present to maintain pH during the normal shelf life of theproduct where less than 10% degradation will take place.

The presence of moisture in the product will have a negative impact onproduct stability. It is therefore preferred that the formulation havethe level of moisture as low as possible during the processing and tomaintain that level throughout the shelf life of the product.

The rate of freezing also has an effect on product stability. The uniquekinetics of the degradation pathway indicates that it is imperative thatthe PGE-1 molecules be kept as far apart as possible in order tominimize the interaction of two PGE-1 molecules. Typically, thelyophilization cycle is designed to proceed as fast as possible withoutexceeding the melting temperature of the frozen solution during primarydrying. Therefore, as long as no meltback occurs during the primarydrying phase and the water content is reduced to a sufficiently lowlevel during the secondary drying phase, then the product would normallybe acceptable. However, the PGE-1 formulation can be quite differentfrom the normal situation because the major component in the formulationis lactose. The literature reports that lactose possesses a very lowglass transition temperature (T_(g)) which is on the order of -31° C. Itis possible to lyophilize above the glass transition temperature for anexcipient such as lactose without exceeding the melting temperature ofthe bulk solution. If the product temperature exceeds T_(g), the frozensolution viscosity decreases significantly resulting in a rubbery systemwhere the mobility of the PGE-1 molecules will increase substantially.This type of event could lead to a situation where the PGE-1 moleculescould either aggregate, micellize, or come into closer proximity than ifthe frozen solution is kept below the T_(g). It is important, therefore,that the drying cycle be optimized to prevent such an occurrence fromhappening.

Typically, the PGE-1 in lactose formulation is freeze dried usingstandard techniques, More preferred, an annealing process is used toenable the residual tertiary butyl alcohol to be reduced and controlled.In an annealing process the initial stage of the freeze drying processis carried out by freezing the PGE-1 formulation to about -50° C.,warming it to about -25° C. for about 2 hours then refreezing it toabout -50° C. Next, the freeze drying is continued to obtain a moisturecontent of less than 1% by dry weight and a tertiary butyl alcoholcontent of less than 3% by dry weight.

The conclusion is that in order to achieve the proper product stability,not only must the formulation be carefully chosen, but also themanufacturing process must be appropriately optimized. The mechanismsmay not be fully understood on a theoretical basis at this time,however, the effects described are reproducible. It is therefore,mandated that a conservative cycle be used to consistently achievemaximum product stability.

Prototype lactose base formulations of PGE-1 indicated that thestability correlated well with an Arrhenius type temperaturerelationship. This fit to an Arrhenius relationship was apparent whetherthe rate constants were plotted for the degradation rate of PGE-1 or forthe rate of formation of the major degradation product PGA₁. Therefore,the stability of the lactose formulation for PGE-1 can be accuratelyassessed by initial rate type kinetic analysis (i.e., by monitoring therate of PGA₁ formation).

Optimization of a freeze-dried formulation PGE-1 (Alprostadil S.Po.) andpreferably as designed for use in an injectable such as in the treatmentof erectile dysfunction has been determined as explained above. Theformulation appears to degrade through an apparent second ordermechanism with respect to PGE-1 concentration in the solid state.Maximum stability can be achieved by either minimizing the PGE-1concentration in the lactose diluent or by optimizing those parameterswhich impact the second order rate constant. The amount of lactosediluent chosen for the optimized formulation was based on solubilitylimitations and the irritation potential of the lactose. In oneembodiment the amount of PGE-1 present was based on the proposedclinical dose for an injection volume of 1 ml or less. The second orderrate constant is affected by the solid state pH, the buffer content, themoisture content, the use of tertiary butyl alcohol during processing,the freezing rate, and the drying rate. All of these have been optimizedto minimize the value of the second order rate constant. The product islyophilized from a bulk sterile filtered solution which contains 20% v/vtertiary butyl alcohol (TBA) and has an apparent pH of approximately 4.Both the water and TBA are removed during the freeze-dying process.Residual water and TBA remaining after lyophilization are<0.5% and 0.5to 2% respectively of the dried cake mass. The final formulation, forexample, per vial contains after completion of lyophilization: 23 μg ofPGE-1 (alprostadil), 193.8 mg of anhydrous lactose, and 53 μg of sodiumcitrate. After reconstitution of this freeze-dried powder with 1.0 ml ofeither water for injection or bacteriostatic water for injection, asolution containing 20 μg/ml of PGE-1 will be obtained. Or, the finalformulation, for example, per vial contains after completion oflyophilization: 11.9 μg of PGE-1 (alprostadil), 193.8 mg of anhydrouslactose, and 53 μg of sodium citrate. After reconstitution of thisfreeze-dried powder with 1.0 ml of either water for injection orbacteriostatic water for injection, a solution containing 10 μg/ml ofPGE-1 will be obtained. Or, the final formulation, for example, per vialcontains after completion of lyophilization: 6.1 μg of PGE-1(alprostadil), 193.8 mg of anhydrous lactose, and 53 μg of sodiumcitrate. After reconstitution of this freeze-dried powder with 1.0 ml ofeither water for injection or bacteriostatic water for injection, asolution containing 5 μg/ml of PGE-1 will be obtained. The freeze-driedpowder, as per these examples, is packaged in a 5 ml vial, sealed with alyophilization style closure within the freeze-dry chamber, and cappedwith an aluminum overseal. The chemical stability of the PGE-1 can bepredicted by use of the Arrhenius equation and accelerated stabilitydata. Initial rate kinetic analyses (i.e., monitoring the rate offormation of the major degradation product, PGA₁) can also be used toassess the chemical stability. The projected stability analysisindicates that when the product is properly manufactured with theoptimized formulation and process, the shelf-life should be greater than24 months when the product is stored at 25° C. or less.

A lot for the various strengths of PGE-1 stabilized product freeze driedunder the conditions described above was prepared and stabilitymeasured. The results are shown in the Tables that follow:

                  TABLE I                                                         ______________________________________                                        20 μg Strength                                                                          Potency at 5° C.                                                                   Potency at 25° C.                             Time (months)                                                                              (% of Initial)                                                                            (% of Initial)                                       ______________________________________                                        0            100.5%      100.5%                                               0            100.0%      100.0%                                               0            100.5%      100.5%                                               0             99.5%      99.5%                                                0            100.0%      100.0%                                               3             99.5%      100.5%                                               3            100.5%      101.5%                                               6            100.0%      98.5%                                                6            --          100.5%                                               9             99.5%      96.6%                                                9             99.0%      98.5%                                                11.44        100.0%      96.6%                                                11.44         99.5%      96.6%                                                11.44         99.5%      96.1%                                                12            99.5%      97.6%                                                12           100.5%      97.6%                                                12           101.0%      97.6%                                                12           101.0%      97.6%                                                15           101.0%      96.1%                                                15           102.0%      95.6%                                                18           100.0%      95.6%                                                18            99.5%      94.6%                                                21            99.0%      96.1%                                                21            99.5%      95.1%                                                ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        10 μg Strength                                                                          Potency at 5° C.                                                                   Potency at 25° C.                             Time (months)                                                                              (% of Initial)                                                                            (% of Initial)                                       ______________________________________                                        0            99.1%        99.1%                                               0            100.9%      100.9%                                               0            100.0%      100.0%                                               0            100.0%      100.0%                                               0             99.1%       99.1%                                               0            101.9%      101.9%                                               0            100.9%      100.9%                                               0            100.9%      100.9%                                               0            100.0%      100.0%                                               0            100.0%      100.0%                                               1             99.1%      102.8%                                               1            100.0%      102.8%                                               1            100.0%      102.8%                                               2            100.9%      102.8%                                               2            102.8%      102.8%                                               2            102.8%      102.8%                                               3             99.1%      100.0%                                               3            100.9%       99.1%                                               3             99.1%       98.1%                                               4            100.9%      100.9%                                               4            100.9%      100.9%                                               4            100.9%      100.0%                                               5            100.0%       97.2%                                               5            100.0%       99.1%                                               5            100.0%       99.1%                                               6             98.1%       97.2%                                               6             97.2%       98.1%                                               6             96.3%       98.1%                                               7.95          97.2%      --                                                   7.95          98.1%      --                                                   7.99          98.1%      --                                                   7.99          98.1%      --                                                   8             98.1%      --                                                   8             97.2%      --                                                   9            101.9%       99.1%                                               9            100.9%       99.1%                                               9            102.8%      100.0%                                               12            98.1%       97.2%                                               12            98.1%       96.3%                                               12            99.1%       96.3%                                               15            99.1%       96.3%                                               15            99.1%       94.3%                                               15            99.1%       95.3%                                               ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        5 μg Strength                                                                           Potency at 5° C.                                                                   Potency at 25° C.                             Time (months)                                                                              (% of Initial)                                                                            (% of Initial)                                       ______________________________________                                        0            101.5%      101.5%                                               0             99.3%      99.3%                                                0            100.4%      100.4%                                               0             98.9%      98.9%                                                0             99.6%      99.6%                                                0             99.8%      99.8%                                                0             99.6%      99.6%                                                0            100.9%      100.9%                                               0            100.2%      100.2%                                               0            100.4%      100.4%                                               1            101.1%      100.7%                                               1            100.7%      99.4%                                                1            100.2%      99.8%                                                2            100.0%      99.3%                                                2            100.6%      100.7%                                               2            100.6%      99.4%                                                3            100.4%      99.8%                                                3            100.4%      100.2%                                               3            100.6%      99.8%                                                4            103.5%      100.0%                                               4             99.6%      99.4%                                                4            100.6%      100.6%                                               5             98.9%      99.4%                                                5            100.0%      99.4%                                                5            100.9%      100.2%                                               6            100.7%      99.4%                                                6            100.9%      99.6%                                                6            100.9%      100.2%                                               6            100.0%      99.1%                                                6             99.3%      98.5%                                                ______________________________________                                    

The Tables show that excellent stability was maintained for the life oflyophilized product.

What is claimed:
 1. A method for preparing a stabilized, lyophilizedformulation of PGE-1 comprising the steps of:a) adding PGE-1 to lactoseand tertiary butyl alcohol wherein said tertiary butyl alcohol ispresent in an amount of from about 15% to about 33% volume/volume andthe ratio of said lactose to PGE-1 is from about 40,000 to 1 to about10,000 to 1 weight/weight whereby a formulation of PGE-1 dispersed inlactose is formed; b) adjusting the pH of said formulation from about 4to about 5 with an organic acid buffer selected from the groupconsisting of citrate and acetate; c) freezing said formulation to about-50° C., warming to about -25° c. for about two hours and thenrefreezint to about -50° C.; and d) drying said formulation to obtain amoisture content of less than 1% by dry weight and a tertiary butylalcohol content of less than 3% by dry weight.
 2. The method of claim 1wherein in said organic acid buffer of step b) is sodium citrate.
 3. Themethod of claim 1 wherein in PGE-1 is in an amount of about 25 to about100 ppm in lactose.